In vitro and in vivo uroselectivity of B8805-033, an antagonist with high affinity at prostatic alpha1A- vs. alpha1B- and alpha1D-adrenoceptors.

We have investigated the pharmacological properties of B8805-033 [(+/-)- 1,3,5-trimethyl-6-[[3-[4-((2,3-dihydro-2-hydroxymethyl)-1,4-benzodioxin-5-yl)-1-piperazinyl]propyl] amino]-2,4(1H,3H)-pyrimidinedione], a new alpha1A-adrenoceptor (AR) selective antagonist. In radioligand binding studies, B8805-033 was 150- to 1200-fold selective for alpha1A-ARs (pKi rat cerebral cortex 8.70, cloned human receptor 7.71) relative to alpha1B-ARs (pKi rat cerebral cortex 5.60, rat liver 5.39, cloned human receptor 5.16) and alpha1D-ARs (pKi cloned human receptor 5.49). B8805-033 inhibited noradrenaline (NA) induced contractions mediated by alpha1A-ARs in rat vas deferens and rabbit and human prostate (pA2 7.62-8.40) much more potently than those mediated by alpha1B-ARs in guinea pig and mouse spleen or by alpha1D-ARs in rat aorta and pulmonary artery (pA2 5.21-5.52). With the exception of a high agonist affinity at 5-HT1A receptors (pKi 9.74 in pig cortex, pD2 6.82 for contraction of rabbit basilar artery) and a moderate to low affinity at histamine H1-receptors (pA2 6.74) and beta1-ARs (pA2 5.75), B8805-033 did not interact with a number of other neurotransmitter receptors (pKi or pA2<5.0). From the i.v. doses of B8805-033 to either inhibit the urethral pressure response to NA by 50% (29 nmol/kg) or to evoke a fall in diastolic blood pressure by 25% (1.54 micromol/kg) in anaesthetized dogs, an urethral/ vascular selectivity ratio of 52 was obtained, far exceeding that found for the nearly unselective prazosin (ratio 1.8). We conclude that B8805-033 is a highly alpha1A-AR selective antagonist, which may potentially be useful as pharmacological tool to investigate alpha1-AR heterogeneity and in the treatment of benign prostatic hyperplasia.

Rhodamine 123 efflux modulation in the presence of low or high serum from CD56+ hematopoietic cells or CD34+ leukemic blasts by B9309-068, a newly designed pyridine derivative.

The newly designed pyridine derivative B9309-068 and a series of structurally different compounds were tested for their ability to modulate rhodamine 123 (RHO) efflux from CD56+ hematopoietic cells in the presence of either 10% fetal calf serum or undiluted human AB serum. Furthermore, efflux modulation was investigated on CD34+ blast populations obtained from four patients with relapsed state AML. Target cells were specified throughout by labeling with peridinine chlorophyll protein (PerCP)-conjugated monoclonal antibodies, allowing clear differentiation from RHO emission spectrum by flow cytometry. In the presence of low serum each compound efficiently modulated RHO efflux without significant differences in the range of final concentrations (1.0-3.0 microM). At 0.1 microM, however, RHO efflux was differentially modulated following the series GF120918 approximately B9309-068 > PSC 833 > DNIG approximately DVER. With CD56+ cells in the presence of undiluted human AB serum at a final modulator concentration of 0.1 microM, all chemosensitizers tested were found to be inefficient. At final concentrations of 0.3 microM or higher, distinct RHO efflux modulation was found with the following efficacies: B9309-068 approximately GF120918 > PSC 833 >> DVER approximately DNIG. The efficacies seen in undiluted human AB serum at 3.0 microM were comparable to those seen on CD56+ cells at final modulator concentrations of 0.1 microM in low serum. Our results identify the pyridine derivative B9309-068 as a promising compound for modulating P-glycoprotein-mediated drug resistance under conditions resembling the clinical setting. Nonetheless, modulation potencies of a series of structurally very different chemosensitizers was revealed to be substantially diminished at high serum concentrations in vitro.

Effects of the selective bisindolylmaleimide protein kinase C inhibitor GF 109203X on P-glycoprotein-mediated multidrug resistance.

Inhibition of protein kinase C (PKC) is discussed as a new approach for overcoming multidrug resistance (MDR) in cancer chemotherapy. For evaluation of this concept we applied the bisindolylmaleimide GF 109203X, which shows a highly selective inhibition of PKC isozymes alpha, beta 1, beta 2, gamma, delta and epsilon in vitro. The efficacy of this compound in modulation of MDR was examined using several P-glycoprotein (P-gp)-overexpressing cell lines including a MDR1-transfected HeLa clone, and was compared with the activities of dexniguldipine-HCI (DNIG) and dexverapamil-HC1 (DVER), both of which essentially act via binding to P-gp. As PKC alpha has been suggested to play a major role in P-gp-mediated MDR, cell lines exhibiting different expression levels of this PKC isozyme were chosen. On crude PKC preparations or in a cellular assay using a cfos(-711)CAT-transfected NIH 3T3 clone, the inhibitory qualities of the bisindolylmaleimide at submicromolar concentrations were demonstrated. At up 1 microM final concentrations of the PKC inhibitor GF 109203X, a concentration at which many PKC isozymes should be blocked substantially, no cytotoxic or MDR-reversing effects whatsoever were seen, as monitored by 72 h tetrazolium-based colorimetric MTT assays or a 90 min rhodamine 123 accumulation assay. Moreover, depletion of PKC alpha by phorbol ester in HeLa-MDR1 transfectants had no influence on rhodamine 123 accumulation after 24 or 48 h. MDR reversal activity of GF 109203X was seen at higher final drug concentrations, however. Remarkably, [3H]vinblastine-sulphate binding competition experiments using P-gp-containing crude membrane preparations demonstrated similar dose dependencies as found for MDR reversion by the three modulators, i.e. decreasing efficacy in the series dexniguldipine-HCl > dexverapamil-HCl > GF 109203X. Similar interaction with the P-gp in the micromolar concentration range was revealed by competition of GF 109203X with photoincorporation of [3H]azidopine into P-gp-containing crude membrane preparations. No significant effect of the PKC inhibitor on MDR1 expression was seen, which was examined by cDNA-PCR. Thus, the bisindolylmaleimide GF 109203X probably influences MDR mostly via direct binding to P-gp. Our work identifies the bisindolylmaleimide GF 109203X as a new type of drug interacting with P-gp directly, but does not support the concept of a major contribution of PKC to a P-gp-associated MDR, at least using the particular cellular model systems and the selective, albeit general, PKC inhibitor GF 109203X.

The leukotriene LTD4 receptor antagonist MK571 specifically modulates MRP associated multidrug resistance.

The multidrug resistant cell lines HL60/AR and GLC4/ADR show high overexpression of the gene encoding the multidrug resistance associated protein MRP compared to their drug sensitive parental counterparts. This and the virtual absence of mdr1/P-glycoprotein gene expression was proven by a complementary DNA polymerase chain reaction (cDNA-PCR) approach. Applying a 72-hour tetrazolium based colorimetric MTT-assay we demonstrate on both MDR sublines a dose-dependent modulation of drug resistances by the leukotriene LTD4 receptor antagonist MK571. A complete reversal of vincristine resistances was achieved at final MK571 concentrations of 30 microM (HL60/AR) or 50 microM (GLC4/ADR) which by itself did not disturb cellular proliferation. The drug resistance of a mdr1/P-gp overexpressing multidrug-resistant HL60 subline, in contrast, was not significantly affected by MK571. Similar effects were seen using the glutathione (GSH) synthesis inhibitor buthionine sulfoximine (BSO). Our results point to a relationship between MRP and a conjugate transporter and identify MK571 as a new tool structure for developing modulators specific for a MRP associated multidrug resistance.

The specific bisindolylmaleimide PKC-inhibitor GF 109203X efficiently modulates MRP-associated multiple drug resistance.

The newly identified drug transporter MRP is functionally linked to a multiple drug resistance independent from P-glycoprotein. Resistance modifiers for this type of MDR are rare at present. We analyzed the modulating effect of the highly selective bisindolylmaleimide PKC inhibitor GF 109203X on the MRP overexpressing human MDR sublines HL60/AR and GLC4/ADR. Applying a 72 hour MTT-assay we demonstrate a complete reversal of the vincristine resistance of HL60/AR cells. Adriamycin resistance of HL60/AR, or vincristine resistance of GLC4/ADR were partially reversed. Furthermore, rhodamine 123 efflux from HL60/AR was strongly modulated by GF 109203X. Since the PKC inhibitor did not significantly influence MRP gene expression at the mRNA level which was examined by cDNA-PCR, our results suggest either a direct interaction of the compound with MRP or/and an indirect influence on MRP activity via altering the phosphorylation status of the transporter.

Vasodilatation elicited by 5-HT1A receptor agonists in constant-pressure-perfused rat kidney is mediated by blockade of alpha 1A-adrenoceptors.

The vasodilator mechanism of the putative serotonin1A (5-HT) receptor agonists, urapidil, 5-methyl-urapidil, ipsapirone, flesinoxan and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was investigated in constant-pressure perfused rat kidneys. The compounds (10(-12)-10(-7) mol bolus injection) neither enhanced basal flow nor evoked vasodilatation in kidneys preconstricted by 27 mM KCl, 1.5 mM BaCl2 or 10(-6) M prostaglandin (PG)F2 alpha, but evoked a dose-dependent, reversible and spiroxatrine-resistant increase in vasodilatation of organs preconstricted by 6 x 10(-7) M noradrenaline. 5-Carboxamidotryptamine and sumatriptan did not reverse the vasoconstriction induced by all stimuli or that induced by noradrenaline in the presence of 5-HT2 plus 5-HT3 receptor blockade. No correlation for the vasorelaxant drugs was found between their -log ED50 in rat kidney and pKi values at 5-HT1A binding sites in pig cortex as determined in radioligand experiments. The relaxation in rat kidney induced by 5-HT1A receptor agonists and alpha 1A-adrenoceptor-selective antagonists (WB 4101 and (+)-niguldipine) was significantly correlated with pKi values at alpha 1A binding sites in rat cortex and the pA2 values derived from contraction studies for competitive antagonism at alpha 1-adrenoceptors in prostatic portions of the rat vas deferens, but differed from pKi values for alpha 1B binding sites in rat cortex. Thus, the vasodilator effect of the 5-HT1A receptor agonists urapidil, 5-methyl-urapidil, ipsapirone, flesinoxan and 8-OH-DPAT in the noradrenaline-perfused rat kidney appears to be mediated by their concomitant alpha 1A-adrenoceptor blockade. No evidence for a vasodilator effect mediated through 5-HT1A receptors was found under our experimental conditions.

Alpha 1-receptor antagonists urapidil and prazosin inhibit neointima formation in rat carotid artery induced by balloon catheter injury.

Sympathetic nerves and catecholamines seem to have a trophic influence on vascular smooth muscle cells in vivo. We therefore tested whether alpha 1-antagonists would inhibit proliferation in arterial smooth muscle in vivo. Smooth muscle cells were stimulated to form a neointima in rat carotid arteries after deendothelialization by means of a 2F embolectomy catheter. The development of intimal lesions was determined 14 days after injury. The size of the neointima was measured using two parameters. Intimal DNA content was estimated from 5-mm segments of carotid arteries after DNA extraction and staining with Hoechst-stain. The size of the neointima was determined morphometrically as intimal area in histological cross-sections. Prazosin and urapidil were given orally once per day. Urapidil-treated rats showed significant inhibition of neointima formation for both parameters in a dose-dependent fashion. For prazosin a significant reduction could only be observed if DNA content was considered.

Stereoselective inhibition of thromboxane-induced coronary vasoconstriction by 1,4-dihydropyridine calcium channel antagonists.

The biological activity of the (+)-S- and (-)-R-enantiomers of niguldipine, of the (-)-S- and (+)-R-enantiomers of felodipine and nitrendipine, and of rac-nisoldipine and rac-nimodipine was investigated in vitro and in vivo. Inhibition of coronary vasoconstriction due to the thromboxane A2 (TxA2)-mimetic U-46619 in guinea pig Langendorff hearts, displacement of (+)-[3H]isradipine from calcium channel binding sites of guinea pig skeletal muscle T-tubule membranes, and blood pressure reduction in spontaneously hypertensive rats were determined. The enantiomers were obtained by stereoselective synthesis. Cross-contamination was less than 0.5% for both S- and R-enantiomers of niguldipine and nitrendipine and less than 1% for those of felodipine. From the doses necessary for a 50% inhibition of coronary vasoconstriction, stereoselectivity ratios for (+)-(S)-/(-)-(R)-niguldipine, (-)-(S)-/(+)-(R)-felodipine, and (-)-(S)-/(+)-(R)-nitrendipine of 28, 13, and 7, respectively, were calculated. The potency ratio rac-nisoldipine/rac-nimodipine was 3.5. Ratios obtained from binding experiments and antihypertensive activity were (+)-(S)-/(-)-(R)-niguldipine = 45 and 35, (-)-(S)-/(+)-(R)-felodipine = 12 and 13, (-)-(S)-/(+)-(R)-nitrendipine = 8 and 8, and rac-nisoldipine/rac-nimodipine = 8 and 7, respectively. Highly significant correlations were found between the in vitro potency of the substances to prevent U-46619-induced coronary vasoconstriction and their affinity for calcium channel binding sites as well as their antihypertensive activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of 5-HT1A receptors in blood pressure reduction by 8-OH-DPAT and urapidil in cats.

This study investigated the effects of (-)-pindolol, a putative 5-HT1A receptor antagonist, upon the central hypotensive action of the antihypertensive drug urapidil and of the purported 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) in cats. Chloralose/urethane-anesthetized cats were thoracotomized and artificially ventilated. Blood pressure was monitored in the iliac artery, and the drugs were injected into the vertebral artery. Urapidil (1-300 nmol/kg) or 8-OH-DPAT (0.01-1 nmol/kg) dose-dependently reduced blood pressure. (-)-Pindolol (30 and 100 nmol/kg) shifted the dose-response curves of both drugs significantly and in a similar manner to the right. Doses of urapidil of 30 nmol/kg or higher also reduced the elevation of blood pressure following the intravenous injection of the alpha 1-adrenoceptor agonist cirazoline whereas 8-OH-DPAT was ineffective. Yet, the hypotensive response to the directly acting vasodilator nitroglycerin remained unchanged after urapidil. The results support the hypothesis that the centrally mediated component of the antihypertensive action of urapidil is due to stimulation of 5-HT1A receptors in the brainstem. Peripheral alpha 1-adrenoceptor blockade comes into play with higher doses of the drug administered via the vertebral artery.

Involvement of brain 5-HT1A receptors in the hypotensive response to urapidil.

Stimulation of serotonin-1A (5-hydroxytryptamine) (5-HT1A) receptors in the brain stem has been suggested to contribute to the antihypertensive action of the alpha 1-adrenoceptor antagonist urapidil. This hypothesis was tested by analyzing the influence of the 5-HT1A receptor antagonist spiroxatrine on the hypotensive responses to urapidil and the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). Chloralose/urethane-anesthetized cats underwent thoracotomy and were artificially ventilated. Blood pressure was monitored in the femoral artery. Urapidil (0.01 to 10 mumol/kg) or 8-OH-DPAT (3 to 30 nmol/kg) was injected into a femoral vein and the maximal hypotensive response recorded. A dose-response test with both drugs was performed before and after administration of spiroxatrine (3 and 10 nmol/kg); the latter was given through the vertebral artery, thus delivering the antagonist to the brain stem. Blood pressure was dose-dependently reduced by urapidil and 8-OH-DPAT after intravenous injection. Central administration of spiroxatrine through the vertebral artery shifted the dose-response curves of both drugs markedly and in a dose-dependent manner to the right, while the hypotensive response to the peripheral vasodilator nitroglycerin remained unchanged. The results suggest that the hypotensive response after peripheral administration of urapidil is mediated in part by stimulation of brain 5-HT1A receptors and this effect on central cardiovascular regulation is additive to the blood pressure reduction resulting from peripheral alpha-adrenoceptor blockade.

Evidence for the interaction of urapidil with 5-HT1A receptors in the brain leading to a decrease in blood pressure.

Current knowledge about the role of serotonin (5-HT) in central cardiovascular regulation is reviewed. Results from experiments with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) suggest that activation of somatodendritic 5-HT1A receptors in the medulla oblongata decreases the firing of serotoninergic neurons and thus reduces their excitatory input to the sympathetic neurons in the intermediolateral cell column. As a consequence, blood pressure is reduced by 5-HT1A receptor agonists. Urapidil is an antihypertensive drug that has a dual mode of action: peripheral alpha-adrenoceptor antagonism and interaction with 5-HT1A receptors in the brain. This profile can adequately explain the vasodilation and lack of significant sympathetic activation observed during urapidil treatment.

Interaction of urapidil with brain serotonin-1A receptors increases the blood pressure reduction due to peripheral alpha-adrenoceptor inhibition.

The alpha-adrenoceptor antagonist urapidil influences central cardiovascular regulation, and this effect is unrelated to alpha-adrenoceptors. Since urapidil has appreciable affinity and selectivity for serotonin-1A (5HT1A) receptors, the activity of urapidil at these sites may be relevant for the centrally mediated component of its antihypertensive action. The latter hypothesis was tested by analysing the influence of the 5HT1A receptor antagonist spiroxatrine on the hypotensive response to urapidil, in comparison with the influence on the hypotensive response to the 5HT1A receptor agonist 8-OH-DPAT (8-hydroxy-2-[di-n-propylamino]tetralin). Anaesthetized cats were thoracotomized and artificially ventilated. Blood pressure was monitored in the descending aorta, and the drugs were injected into the vertebral artery. Spiroxatrine (0.1-3.0 nmol/kg) shifted the cumulative dose response curve (blood pressure reduction) of urapidil (3-20 nmol/kg) and of 8-OH-DPAT (0.01-0.1 nmol/kg) to the right, suggesting competitive antagonism. The results support the hypothesis that the effects of urapidil on central cardiovascular regulation and at least part of the hypotensive effects are due to 5HT1A receptor stimulation.

Mechanoreceptors in rat glabrous skin: redevelopment of function after nerve crush.

In the glabrous skin of the rat's hindfoot the same triple set of low-threshold mechanoreceptors is present as has been found in other mammals: slowly adapting (SA), rapidly adapting (RA), and very rapidly adapting Pacinian corpuscle-like (PC) receptors. Their functional characteristics were examined in normal rats and compared with those of sensitive mechanoreceptors found in the glabrous skin of the foot 2-24 wk after crush of the plantar nerves, resulting in regeneration of the transected nerve fibers. After 2 wk of nerve regeneration, low-threshold RA and SA cutaneous mechanoreceptors reappeared in the foot skin. Responses of PC receptors were recorded again after 3 wk, at which time the proportion of fibers that could be identified as low-threshold cutaneous mechanoreceptors had regained control level. Discharge patterns of regenerated cutaneous mechanosensitive receptors were very similar to those of normal skin mechanoreceptors. Their sensitivity to controlled mechanical stimulation was, however, still reduced 4 wk after the lesion. After 8 wk RA and SA receptors had regained their normal dynamic sensitivity, i.e., the responsiveness to the velocity of skin indentation. The static sensitivity of SA receptors, i.e., responsiveness to maintained skin indentation, was not consistently reestablished within 24 wk. No shift in sensitivity could be deduced from tuning curves of PC receptors examined 3-24 wk after nerve crush. In addition to the low-threshold mechanoreceptors, high-threshold (HT) mechanoreceptive fibers were found in controls and in animals with regenerating nerves. This type of fiber was most frequently found 1 wk after the nerve crush, when reinnervation of the foot started. They probably represent fibers not connected to specific mechanoreceptor end organs. Thus, functional restitution of the highly specific cutaneous mechanoreceptors occurs fairly soon after invasion of the original territory by the regenerating nerve. It is assumed that the underlying mechanism is the rapid reconnection of fibers with the end organs that have either survived during the period of denervation or regenerated subsequent to reinnervation of the skin.

Influence of urapidil on alpha- and beta-adrenoreceptors in pithed rats.

The interaction of urapidil with pre- and postsynaptic alpha-adrenoreceptors and with postsynaptic beta-adrenoreceptors was studied in pithed normotensive rats and compared to the effects of clonidine, prazosin, and atenolol. I.v. injection of urapidil did not substantially change blood pressure, while clonidine raised blood pressure. Urapidil dose-dependently antagonized the pressor effects of the alpha 1-agonist L-phenylephrine (pDR2 6.8) and of the alpha 2-agonist azepexole (pDR2 5.2). Compared to urapidil, prazosin was a more potent antagonist of phenylephrine at postsynaptic vascular alpha 1-adrenoreceptors (pDR2 8.7) and of azepexole at alpha 2-adrenoreceptors (pDR2 5.6). Urapidil inhibited the tachycardia produced by discontinuous or continuous electrical stimulation of the thoracic spinal outflows (ID50 4.8 and 27.2 mumol/kg, respectively). In contrast to the inhibitory action of clonidine (ID50 0.039 and 0.023 mumol/kg, respectively), the inhibition by urapidil was not reversed by the selective alpha 2-antagonist rauwolscine (10 mumol/kg). Prazosin did not change stimulation-evoked tachycardia but atenolol caused pronounced inhibition (ID50 0.158 mumol/kg, discontinuous stimulation). Urapidil dose-dependently antagonized the tachycardic effect of isoprenaline at beta 1-adrenoreceptors (pDR2 5.1) but also exhibited intrinsic activity by increasing basal heart rate (maximum effect of urapidil was 30% of that of isoprenaline). Urapidil did not change the vasodilatory beta 2-adrenoreceptor-mediated effect of isoprenaline. The results suggest that urapidil is an antagonist at postsynaptic vascular alpha 1- and alpha 2-adrenoreceptors, with a greater potency against alpha 1-adrenoreceptors. An agonistic interaction of urapidil with presynaptic alpha 2-adrenoreceptors could not be demonstrated in pithed rats. Instead, the inhibition by urapidil of stimulation-evoked tachycardia could be accounted for by its beta 1-adrenoreceptor antagonistic effect.

Effects of urapidil, clonidine, prazosin and propranolol on autonomic nerve activity, blood pressure and heart rate in anaesthetized rats and cats.

The influence of urapidil, clonidine, prazosin and propranolol on autonomic nerve activity was determined in anaesthetized cats and rats. The effects of these drugs on blood pressure and heart rate were also evaluated. Impulse output was recorded in the splanchnic and vagus nerve of the cat, and in the cervical sympathetic trunk of the rat. Urapidil increased activity in sympathetic and parasympathetic nerve fibres in cats at low doses without affecting blood pressure and heart rate. At higher doses which lowered blood pressure, urapidil reduced sympathetic impulse output in cats and rats while vagal output was increased. The alpha 1-adrenoceptor blocking agent, prazosin, did not affect activity in sympathetic and parasympathetic nerve fibres while the beta-adrenoceptor blocking agent, propranolol, increased activity in these nerves in cats. The alpha-adrenoceptor agonist, clonidine, reduced sympathetic impulse output at all doses tested in both rats and cats. The results provide evidence that urapidil, in addition to its peripheral alpha- and beta-adrenoceptor blocking properties, affects cardiovascular regulation by a central action. Blockade of alpha- or beta-adrenoceptors in the brain is probably not responsible for the central effect of urapidil.

Biotransformation of urapidil: metabolites in serum and urine and their biological activity in vitro and in vivo.

The biotransformation products of the antihypertensive drug urapidil were determined in the serum and urine of rat, dog and man. Comparison of the pattern of urapidil metabolism revealed that all species examined formed the same metabolites. However, quantitative differences were noted. The pattern of metabolites in serum was seen to parallel the excretion of urapidil and its metabolites in urine. The p-hydroxylated urapidil (M1) was found to be the predominant metabolite in man, the uracil-N-demethylated metabolite (M3) in dog, and M1 as well as the O-demethylated urapidil (M2) were found to be important in the rat. Furthermore, trace amounts of the urapidil-N-oxide (M5) are found in dog urine. As determined in the isolated rat vas deferens preparation, urapidil and the synthetized metabolites M1, M2, M3 and M5 are competitive antagonists of the effects of noradrenaline at postsynaptic alpha 1-adrenoceptors. Metabolites M2 and M3 exhibit alpha 1-adrenoceptor antagonism (pA2 values of 6.79 and 6.93 respectively) which are comparable to urapidil (pA2 = 7.02), whilst M1 and M5 are less potent antagonists giving pA2 values of 5.69 and 5.55 respectively. On i.v. or i.j. administration of urapidil, M1, M2, M3 or M5 to anaesthetized, normotensive rats, or orally to conscious spontaneously hypertensive rats, differences in cardiovascular responses were seen. Whilst M1 had little antihypertensive activity, M2 and M3 lowered blood pressure to a similar extent to that seen with urapidil. However, the duration of activity observed was shorter. Urapidil-N-oxide (M5) appeared to possess hypotensive activity comparable to urapidil, but examination of serum samples following the administration of M5 yielded mainly urapidil, suggesting that M5 exhibits its activity following reduction back to urapidil. In view of the serum levels attained and of the biological activity, the metabolites of urapidil do not appear to contribute significantly to the cardiovascular effects of urapidil in man or rat. However, in view of the serum levels and duration of M3 observed in dogs, this metabolite may make a significant contribution to the hypotensive activity of urapidil in this species.

Differential effects of noxious and non-noxious input on neurones according to location in ventral periaqueductal grey or dorsal raphe nucleus.

Nociceptive and non-nociceptive input to the dorsal raphe nucleus (DR) and to the surrounding periaqueductal grey (PAG) was studied in chloralose-anaesthetized rats. Single units in the midbrain responding to electrical stimulation of a coccygeal nerve were recorded with glass micropipettes. A fluorescence histochemical technique was applied to identify recording sites in the DR and PAG. 109 DR-units, 141 PAG-units and 95 units from surrounding structures were tested for responsiveness to electrical nerve stimulation. In 53% of the DR-units, but in only 20% of the PAG- and SN-units, ongoing activity was inhibited by electrical stimulation (I-units) while 42% of the PAG- and SN-units but only 24% of the DR-units were electrically excited (E-units). 40 E-units and 24 I-units were tested with repeated noxious radiant heat stimuli applied to the tail or hindpaws. 70% of the E-units were excited by heating, and in 54% of the I-units ongoing activity was inhibited by heating. The majority of the former units were located in the PAG, and most of the latter were proven to be DR-neurones. In 75% of the E-units and in 12.5% of the I-units the heat effect was in the opposite direction. The findings are discussed in terms of the now well-established role of the PAG-region in the descending control of pain. The properties of the PAG-E-units suggest that this system is involved in a negative feedback circuit by which pain transmission to the CNS limits itself. DR-I-units may be involved via an additional small loop with the PAG to disinhibit the activation of the PAG pain control system.